Drosophila melanogaster, D. simulans and D. mauritiana are closely related sibling species which are similar in morphology and polytene chromosome banding pattern; they can be cross-mated in the laboratory. Nevertheless, several lines of evidence indicate that the genome of D. melanogaster contains a large, single-copy component not present in D. simulans or D. mauritiana. This component may comprise as much as 35% of the total single-copy DNA of D. melanogaster. The objective of the research proposed here is to clone and characterize sequences in this cryptic genomic component, in order to explore its organization, evolution and expression. The approach is to construct a collection of short, random genomic clones from D. melanogaster and screen them in several ways to identify those which are single-copy and absent from D. simulans and/or D. mauritiana. When such clones have been isolated, they will be characterized with respect to chromosomal location (by in situ hybridization to polytene chromosomes), presence in other strains and species (by Southern blotting), transcription during development (by Northern blotting) and DNA sequence. These clones will be used to isolate long genomic clones from a Lambda phage library of D. melanogaster DNA. The Lambda clones will be searched for organizational features which might elucidate the mechanism of interspecies variability. The hypothesis that there is a mechanistic relationship between interspecies variability of single-copy DNA content and the genomic movement of repetitive transposable elements will be tested. Observation that closely related species do not share a major genomic component is unexpected and interesting. The experiments proposed here will provide a start in understanding the evolutionary and physiological implications of this finding.